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Technical Briefs |
-Chain Gene Rearrangements: Prediction of Malignant Transformation of Cutaneous T-Cell Lymphoproliferative Disorders
1 Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, Singapore 119074
2 Department of Pathology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260
aaddress correspondence to this author at: Department of Pathology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260; fax 65-67751757, e-mail patkoaye{at}nus.edu.sg
T-Cell lymphoproliferative disorders of the skin constitute in themselves a unique area of diagnostic difficulty for the clinician and the pathologist. Hence, although T-cell clonality analysis by PCR and capillary electrophoresis (CE) has been used extensively in lymphoproliferative disorders in general, there are very few reports describing their application to the differential diagnosis of cutaneous T-cell lymphoma (1)(2) or to cases of cutaneous lymphoproliferative disorders with less conclusive histology, where their superior resolution properties have provided strong confirmatory evidence of evolving lymphoma development (3).
We present here our validation data of the use of CE in the analysis of T-cell receptor 
-chain (TCR) gene rearrangements for clinical use. We also present a report of a case that was judged to be benign based on clinical and pathologic grounds but was found to have the clonal molecular fingerprint that could have predicted malignant transformation at an early stage. This predictive value, in our opinion, enhances the diagnostic value of this test in the clinical setting.
Pathologists are aware that distinction between benign and malignant T-cell lymphoproliferative disease by use of morphologic criteria can often be difficult, even with the aid of immunotyping. The best marker of T-cell malignancy is T-lymphocyte monoclonality, which is rarely encountered in nonneoplastic disease. Rearrangements of TCR genes in a rapidly proliferating neoplastic lymphoid cell population derived from a single progenitor cell are identical, involving unique combinations of specific variable (V), joining (J), diversity (D), and constant (C) gene segments, and thus serve as unique clonal markers (3)(4). In contrast, nonneoplastic and reactively proliferating T-cells yield different, polyclonal TCR gene rearrangements.
TCR gene rearrangements can be detected by Southern blotting analysis, which is highly specific by virtue of the labeled hybridization probes (5)(6), but this method is laborious and time-consuming and requires a substantial amount of DNA from either fresh or frozen tissue, which is not always available from biopsies. The application of PCR amplification in the detection of TCR gene rearrangements has been demonstrated to be superior to Southern blotting for routine diagnostic purposes (7)(8). The limited number of V and J gene segments and the absence of D gene segments in the TCR gene make the amplification of this gene much simpler than amplification of the TCRß gene.
The use of CE has recently been advocated for the study of clonality in lymphoid processes (9)(10)(11). CE is superior to gel electrophoresis in that it allows separation and identification of PCR products differing in size by only 1 bp (12). The use of CE also permits more precise determination of the product size without the problems of band-shift artifacts and gel-to-gel variation often associated with agarose or polyacrylamide gel electrophoresis. This is particularly helpful for monitoring recurrent or minimal residual disease in the same patient over time. CE analysis is much more direct and rapid because it does not involve casting and running gels.
We extracted DNA from 15- to 20-µm paraffin-embedded archived (patient consent was obtained at the time of surgery to allow excess sample to be used for research purposes, per hospital Institutional Review Board guidelines) tissue sections of skin or lymph node specimens, using the DNeasy Tissue Kit (Qiagen GmbH). The V and J regions of the TCR gene were amplified using two V-region primers, V11 (5'-TCT GG(G/A) GTC TAT TAC TGT GC-3') and V101 (5'-CTC ACA CTC (C/T)CA CTT C-3') and the 5'-end 5-carboxyfluorescein (FAM)-labeled J-region primer, J (5'-CAA GTG TTG TTC CAC TGC C-3') (13). PCR was carried out with 
100 ng of genomic DNA, 1x PCR AmpliTaq Gold buffer, 5 mM deoxynucleotide triphosphate mixture, 10 µM each of primers V11 and V101, 1 µM primer J, 37.5 mM MgCl2, and 1.5 U of AmpliTaq Gold DNA polymerase. The denaturing, annealing, and extension steps were performed at 94 °C for 45 s, 59 °C for 90 s, and 73 °C for 2 min, respectively, for 35 cycles on a Hybaid PCR Express thermocycler. The PCR protocol also included an initial denaturation step (3 min at 94 °C) and a final extension step (5 min at 72 °C). After PCR, 1.0 µL of the amplified product was added to 12 µL of Hi-diformamide (Applied Biosystems) and 0.5 µL of internal size standard (GeneScanTM 350-ROX; Applied Biosystems), denatured for 2 min at 95 °C, cooled on ice, and injected into an automated ABI PRISM® 310 Genetic Analyzer. Fragment size analysis was determined using the GeneScan software, Ver. 3.7 (Applied Biosystems).
Twenty samples of paraffin-embedded tissue from seven patients with T-lymphoproliferative skin disorders were analyzed. Fourteen samples (10 skin and 4 lymph node samples) had histologic and immunohistochemical evidence of malignancy (T-cell lymphomas), whereas 4 were benign lesions, despite the fact that they were from patients with T-cell lymphomas. Interestingly, two of these lesions with a benign phenotype (parapsoriasis en plaque and dermatitis) were taken from a patient who was diagnosed with T-cell lymphoma 8–9 years later. In addition to these benign lesions, which served as polyclonal controls for the study, we included known cases of lymph node lymphoma (n = 2) and reactive lymphoid changes in lymph nodes (n = 2) in our study, running them in parallel with the study cases. Both of the control cases with an established diagnosis of T-cell lymphoma gave clear monoclonal profiles; the two reactive cases gave polyclonal patterns. Sensitivity studies with serial dilutions of the SUP-T1 cell line (American Type Culture Collection) in placental DNA revealed a detection threshold of 10-2 (equivalent to detection of 1 ng of clonal DNA in a background of 100 ng of placenta DNA).
All 14 samples with clinical, histologic, and immunohistochemical evidence of T-cell lymphoma showed either a single, monoclonal peak or a very prominent monoclonal peak in a polyclonal background. Three of the four samples that were clearly phenotypically benign showed a polyclonal pattern, whereas one (perivascular dermatitis) showed an oligoclonal pattern, in keeping with the presence of circulating malignant cells in the biopsy. There thus was a 100% concordance rate between the well-established clinicopathologic diagnoses and the molecular fingerprints of the diseases. Table 1
shows a summary of the clinical diagnoses vs the GeneScan profiles of the 20 samples studied.
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The most interesting of our findings came from the case (case 5 in Table 1
) with two separate lesions exhibiting benign phenotypes (dermatitis and parapsoriasis en plaque; see Fig. 1
). Approximately 8 years after the second sample was taken, the patient was again diagnosed and found to have angiocentric T-cell lymphoma. The CE GeneScan profiles (Fig. 1
) showed that DNA extracted from both these phenotypically benign lesions had a detectable monoclonal peak, indicating the presence of a clonal T-cell population. Moreover, these monoclonal peaks were identical in base pair size to the diagnostic monoclonal peak detected in the sample that established T-cell malignancy 8 years later.
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This study validates the usefulness of TCR gene rearrangements in the differentiation of benign vs malignant cutaneous T-cell lymphoproliferative disorders. Furthermore, it illustrates the potential of the method to detect a clonal genetic fingerprint in cutaneous lesions that, at the time of presentation, do not show unequivocal clinical or pathologic signs of future malignant transformation. The diagnosis and monitoring of T-cell cutaneous lymphoproliferative disorders with a molecular approach similar to the one validated by us may have important diagnostic, prognostic, and therapeutic implications.
Acknowledgments
This work was funded by grants from the National Medical Research Council (NMRC 0383/1999) and the Health Services Development Department, Ministry of Health Singapore (HSDP01N02). We are grateful to Tan Tee Chok for the photography for Fig. 1
.
References
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[Order article via Infotrieve]The following articles in journals at HighWire Press have cited this article:
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